The present invention relates to polypeptides which stimulate bone growth.
Understanding of issues related to bone growth and strength has progressed over the years, a summary being provided in, for example, international patent application No. PCT/CA 94/00144, published on Sep. 15, 1994 under WO 94/20615, U.S. Pat. No. 5,320,970 and European patent application No. 92 302 446, published under 505 210 on Sep. 23, 1992, the contents of which applications are incorporated herein by reference.
By way of background to the present invention, described below, neutrophil-activating peptide (NAP-2; SEQ ID NO:1) and a variant of NAP-2, here termed xe2x80x9cNAP-2Vxe2x80x9d (SEQ ID NO:2) have been known for some time (Walz, A., and M. Baggiolini, (1989) Biochem. Biophys. Res. Commun. 159:969). British Patent No.2 231 872 (British Patent No. 2 231 872. Inventors: M. Baggiolini, K. J. Clemetson, and A. Walz. Published Jun. 14, 1990.), describes the amino acid sequence of NAP-2 and three apparently naturally occurring variants, including NAP-2V. The other two variants have an additional four (SEQ ID NO:3) and three (SEQ ID NO:4) amino acids at the N-terminal of the NAP-2 sequence. NAP-2 is a subsequence of xcex2-thromboglobulin (xcex2-TG; SEQ ID NO:5) which has an additional eleven amino acids at the N-terminal end. xcex2-TG is itself a subsequence of connective tissue-activating peptide (CTAP-III; SEQ ID NO:6) which has an additional four amino acids at the N-terminal. CTAP-III is a subsequence of platelet basic protein (PBP; SEQ ID NO:7) which has an additional nine amino acids at the N-terminal.
NAP-2 along with interleukin-8 (human IL-8; SEQ ID NO:8; porcine IL-8 SEQ ID NO:9) and melanoma growth-stimulating activity (MGSA) have been assigned to a subfamily known as the xcex1-chemokines. The xcex1-chemokines have in common with each other four cysteine residues at highly conserved positions, which enclose the core region of the molecules, as described by Brandt et al (Ehlert, J. E., F. Peterson, M. H. G. Kubbuta, J. Gerdes, H.-D. Flad, and E. Brandt, (1995) J. Biol. Chem. 270:6338). Brandt et al. found an apparently naturally occurring C-terminus truncated variant of NAP-2, lacking the last four amino acids of NAP-2, that displays enhanced increase in potency to stimulate neutrophil degranulation. Brandt et al. also synthesized variants lacking the final one, two, three, five and six amino acids of the C-terminus of NAP-2. All of these C-truncated polypeptides exhibited a moderate increase in potency over NAP-2 with the exception of the sequence having only the first sixty-four amino acids of NAP-2. Brandt et al. discussed the possible significance of the sequence modifications with respect to the structure of NAP-2 and its function.
Platelet factor 4 (PF4; SEQ ID NO:10) is a seventy amino acid polypeptide (Hermodson, M., G. Schmer and K. Kurachi, (1977) J. Biol. Chem. 252:6276; Morgan, F. J., G. S. Begg, C. N. Chesterman, (1979) Thromb. Haemost 42:1652). PF4 has been shown to inhibit proliferation of two osteoblastic osteosarcoma cell lines, Saos-2 and G-292 (U.S. Pat. No. 5,304,542. Inventor: D. M. Tatakis. Issued Apr. 19, 1994). Indomethacin apparently did not affect PF4-induced inhibition of the cell proliferation. Particular fragments, PF4(58-70), PF4(47-70) and monomeric low-affinity PF4 (LAPF4), which is 50% homologous to PF4 and contains an xcex1-helical C-terminus were also suggested as being useful. PF4 and such related polypeptides were thus described as being useful in a method for inhibiting proliferation of osteoblasts, in among other things, humans suffering from osteoporosis.
The first 70 amino acids of NAP-2V and the sequence of PF4 are about 51% homologous and the positions of the four cysteine residues are conserved between the two polypeptides.
It has now been shown that NAP-2, NAP-2V, as well as subsequences of NAP-2V also show bone stimulatory effects, while certain subsequences do not display bone stimulatory activity. NAP-2V-(1-26) (SEQ ID NO:11) and NAP-2V-(13-26; gln25xe2x86x92glu25) (SEQ ID NO:12) were found to increase the observed bone apposition rate, the latter of the two being more potent than the former. NAP-2V-(10-26) (SEQ ID NO:13) appeared to cause a small increase in the observed bone apposition rate, although the statistical significance of the observed increase was questionable. Protected versions of NAP-2V-(13-26; gln25xe2x86x92glu25) (SEQ ID NO:17) and of NAP-2V-(15-22) (SEQ ID NO:18) were also found to have bond stimulatory activity. NAP-2V-(11-26) (SEQ ID NO:14) and NAP-2V-(12-26) (SEQ ID NO:15) were found to have no effect on observed bone mineral apposition rate.
The invention thus includes a compound which promotes bone growth in mammals, comprising an amino acid sequence which consists essentially of:
(i) up to 69 consecutive amino acids of the sequence corresponding to SEQ ID NO:2 with (a) from 6 to about 14 amino acids deleted from the N-terminus of SEQ ID NO:2, or (b) 7 to about 53 amino acids deleted from the C-terminus of SEQ ID NO:2, or both (a) and (b) and wherein the sequence includes no cysteine residues or at least two cysteine residues;
(ii) a variant of a polypeptide of (i) containing a plurality of said amino acid sequences;
(iii) a conservatively substituted variant of a polypeptide of (i) or (ii); or
(iv) a functionally equivalent homologue of (i), (ii) or (iii).
In another aspect, the invention includes a polypeptide in which the amino acid sequence consists essentially of an amino acid sequence corresponding to SEQ ID NO:11, or corresponding to SEQ ID NO:11 with from 6 to about 14 amino acids deleted from the N-terminus of SEQ ID NO:11, or corresponding to SEQ ID NO:11 with from 6 to about 12 amino acids deleted from the N-terminus of SEQ ID NO:11; or corresponding to SEQ ID NO:11 with from 6 to about 9 amino acids deleted from the N-terminus of SEQ ID NO:11; or a functionally equivalent homologue any of the foregoing polypeptides.
A polypeptide of the present invention can have an amino acid sequence which consists essentially of an amino acid sequence corresponding to SEQ ID NO:12, or SEQ ID NO:13, or SEQ ID NO:18, or a functionally equivalent homologue of any of these.
The present invention includes a compound xe2x80x9cderived fromxe2x80x9d any of the preceding polypeptides.
The present invention thus includes a polypeptide wherein the amino acid sequence consists essentially of:
(v) an amino acid sequence corresponding to SEQ ID NO:12;
(vi) an amino acid sequence corresponding to SEQ ID NO:13;
(vii) an amino acid sequence corresponding to SEQ ID NO:18;
(viii) a variant of a polypeptide of (v), (vi) or (vii) containing a plurality of said amino acid sequences; or
(ix) a conservatively substituted variant of a polypeptide of (v), (vi), (vii) or (viii).
In preferred embodiments, a polypeptide of the present invention that cysteine residues, includes two cysteine residues that are located at positions corresponding to the tenth and twelfth positions of SEQ ID NO:2.
A polypeptide of the present invention can have, if desired, or necessary, one or the other or both of the N-terminal amino acid and the C-terminal amino acid protected by a protecting group.
The polypeptide can be synthetic and the amino acid sequence can have a molecular weight in the range of from about 1000 to 4000 daltons; or from about 1000 to about 3000; or from about 1000 to about 2000; or more preferably from about 1200 to about 1800.
In another aspect, the invention is a first polypeptide which promotes bone growth in mammals comprising a sequence of amino acids which consists essentially of up to 69 amino acids and is sufficiently duplicative of a second polypeptide such that the first polypeptide is encoded by a DNA that selectively hybridizes under stringent conditions with DNA encoding the second polypeptide. In this instance, the second polypeptide comprises an amino acid sequence which consists essentially of:
(i) up to 69 consecutive amino acids of the sequence corresponding to SEQ ID NO:2 with (a) from 6 to about 14 amino acids deleted from the N-terminus of SEQ ID NO:2, or (b) 7 to about 53 amino acids deleted from the C-terminus of SEQ ID NO:2, or both (a) and (b) and wherein the sequence includes no cysteine residues or at least two cysteine residues;
(ii) a variant of a polypeptide of (i) containing a plurality of said amino acid sequences;
(iii) a conservatively substituted variant of a polypeptide of (i) or (ii); or
(iv) a functionally equivalent homologue of (i), (ii) or (iii).
The DNA sequence of NAP-2V disclosed by Walz et al. (British Patent No. 2 231 872. Inventors: M. Baggiolini, K. J. Clemetson, and A. Walz. Published Jun. 14, 1990. Neutrophil-activating peptide-2 and processes for the production of NAP-2, B-TG, CTAP-III and PBP) is identified here as SEQ ID NO:16.
The phrase xe2x80x9cselectively hybridizes toxe2x80x9d refers to a nucleic acid molecules that, under appropriately stringent hybridization conditions, hybridize, duplex or bind essentially only to each other when molecules having the predefined sequences (i.e., second polypeptide) are present in a preparation of DNA or RNA. For discussions of nucleic acid molecule design and annealing conditions, see, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual (2nd ed.), Vols. 1-3, Cold Spring Harbor Laboratory, (1989) or Current Protocols in Molecular Biology, F. Ausubel et al., (ed.) Greene Publishing and Wiley-Interscience, New York (1987).
It will, of course, be understood by those skilled in the art that portions of the nucleic acid sequence identified as SEQ ID NO:16 correspond to sequences coding for the polypeptides identified as SEQ ID NO:1, NO:11; NO:12 (Gluxe2x86x92Gln) or NO:13. For example, nucleic acids 37 through 78 of SEQ ID NO:16 encode the amino acid subsequence identified as SEQ ID NO:12 in which the penultimate amino acid of the subsequence is glutamine.
xe2x80x9cStringent hybridization conditionsxe2x80x9d takes on its common meaning to a person skilled in the art here. Appropriate stringency conditions which promote nucleic acid hybridization, for example, 6xc3x97sodium chloride/sodium citrate (SSC) at about 45xc2x0 C. are known to those skilled in the art. The following examples are found in Current Protocols in Molecular Biology, John Wiley and Sons, NY (1989), 6.3.1-6.3.6: For 50 ml of a first suitable hybridization solution, mix together 24 ml formamide, 12 ml 20xc3x97SSC, 0.5 ml 2 M Tris-HCl pH 7.6, 0.5 ml 100xc3x97Denhardt""s solution, 2.5 ml deionized H2O, 10 ml 50% dextran sulfate, and 0.5 ml 10% SDS. A second suitable hybridization solution can be 1% crystalline BSA (fraction V), 1 mM EDTA, 0.5 M Na2HPO4 pH 7.2, 7% SDS. The salt concentration in the wash step can be selected from a low stringency of about 2xc3x97SSC at 50xc2x0 C. to a high stringency of about 0.2xc3x97SSC at 50xc2x0 C. Both of these wash solutions may contain 0.1% SDS. In addition, the temperature in the wash step can be increased from low stringency conditions at room temperature, about 22xc2x0 C. to high stringency conditions, at about 65xc2x0 C. The cited reference gives more detail, but appropriate wash stringency depends on degree of homology and length of probe. If homology is 100%, a high temperature (65xc2x0 C. to 75xc2x0 C.) may be used. If homology is low, lower wash temperatures must be used. However, if the probe is very short ( less than 100 bp), lower temperatures must be used even with 100% homology. In general, one starts washing at low temperatures (37xc2x0 C. to 40xc2x0 C.), and raises the temperature by 3-5xc2x0 C. intervals until background is low enough not to be a major factor in autoradiography.
Alternatively, such a first polypeptide is a sequence of amino acids sufficiently duplicative of a second polypeptide having an amino acid sequence corresponding to SEQ ID NO:11 up to 69 amino acids in length, or corresponding to SEQ ID NO:11 with from 6 to about 12 amino acids deleted from the N-terminus of SEQ ID NO:11, or corresponding to SEQ ID NO:11 with from 6 to about 9 amino acids deleted from the N-terminus of SEQ ID NO:11; wherein the sequence includes no cysteine residues or at least two cysteine residues; or a functionally equivalent homologue, such that the first polypeptide is encoded by a DNA that hybridizes under stringent conditions with DNA encoding the second polypeptide. The second polypeptide can have, alternatively, or additionally 7 to about 53 amino acids deleted from the C-terminus of SEQ ID NO:2.
The first polypeptide can have a sequence of amino acids sufficiently duplicative of a second polypeptide having an amino acid sequence corresponding to SEQ ID NO:12 up to 69 amino acids in length; or a functionally equivalent homologue, such that the first polypeptide is encoded by a DNA that hybridizes under stringent conditions with DNA encoding the second polypeptide.
The first polypeptide can have a sequence of amino acids sufficiently duplicative of a second polypeptide having an amino acid sequence corresponding to SEQ ID NO:11; or a conservatively substituted variant thereof, such that the first polypeptide is encoded by a DNA that hybridizes under stringent conditions with DNA encoding the second polypeptide; or it can have a sequence of amino acids sufficiently duplicative of a second polypeptide comprising an amino acid sequence corresponding to SEQ ID NO:12; or a conservatively substituted variant thereof, such that the first polypeptide is encoded by a DNA that hybridizes under stringent conditions with DNA encoding the second polypeptide; or it can have a sequence of amino acids sufficiently duplicative of a second polypeptide having an amino acid sequence corresponding to SEQ ID NO:13; or a conservatively substituted variant thereof, such that the first polypeptide is encoded by a DNA that hybridizes under stringent conditions with DNA encoding the second polypeptide; or it can have a sequence of amino acids sufficiently duplicative of a second polypeptide having an amino acid sequence corresponding to SEQ ID NO:18; or a conservatively substituted variant thereof, such that the first polypeptide is encoded by a DNA that hybridizes under stringent conditions with DNA encoding the second polypeptide.
A bone growth polypeptide of the invention can be of any suitable length, and particularly can be up to 8 amino acids in length, or up to 14 amino acids in length, or up to 20 amino acids in length, or up to 30 amino acids in length, or up to 40 amino acids in length, or up to 50 amino acids in length, or up to 60 amino acids or more in length.
The invention includes any number of chimeric bone stimulating factors made having an amino acid sequence of polypeptides of the present invention.
In another aspect, the invention is an agent for use in prevention and treatment of a bone reduction related disease which includes any polypeptide or polypeptides of the present invention as an active ingredient.
The invention is, alternatively, a pharmaceutical composition for promoting bone growth, having a therapeutically effective amount of a polypeptide or polypeptides of the present invention.
The invention includes a method of increasing bone growth in a mammal by administering a therapeutically effective amount of a polypeptide having an amino acid sequence of a polypeptide or polypeptides of the present invention.
The invention includes use of a polypeptide or polypeptides of the present invention for the treatment of osteoporosis. Alternatively, the use of a polypeptide or polypeptides can be to promote bone growth in a mammal.
The invention includes use of the polypeptide or polypeptides in the preparation of a medicament for use in promoting bone growth or the treatment of osteoporosis.
The invention includes a diagnostic kit for determining the presence of a polypeptide or polypeptides of the present invention. The kit can includes an antibody to a said polypeptide(s) linked to a reporter system wherein the reporter system produces a detectable response when a predetermined amount of the polypeptide(s) and the antibody are bound together.
The invention includes an antibody synthesized using a polypeptide consisting of an amino acid sequence identified as SEQ ID NO:11; SEQ ID NO:12; or SEQ ID NO:13 or a conservatively substituted variant thereof.
More generally, the invention includes an antibody which binds to a polypeptide or polypeptides of the present invention, synthesized using the polypeptide(s).
The invention includes an isolated DNA fragment which encodes the expression of any of the polypeptides of the present invention, and DNA which differs from the fragment due to the degeneracy of the genetic code.
The invention includes a vector comprising a DNA sequence which encodes the expression of any of any of the polypeptides of the present invention.
The invention includes a process for producing a polypeptide of the invention which includes the steps of:
a) preparing a DNA fragment containing a nucleotide sequence which encodes the polypeptide;
b) incorporating said DNA fragment into an expression vector to obtain a recombinant DNA fragment which contains the DNA fragment and is capable of undergoing replication;
c) transforming a host cell with the recombinant DNA fragment to isolate a transformant which can express the polypeptide; and
d) culturing the transformant to allow the transformant to produce the polypeptide and recovering the polypeptide from resulting cultured mixture.
In yet another aspect, the invention is a synthetic polypeptide up to 65 amino acids in length, having in vivo bone stimulatory activity in mammals, having an amino acid sequence which is at least about 11% conserved in relation to the amino acid sequence identified as SEQ ID NO:2 (i.e., contains 8 consecutive amino acids of the sequence identified as SEQ ID NO:2); a conservatively substituted variant thereof; or a functionally equivalent homologue.
The synthetic polypeptide can also be at least about 11%, 12%, 13%, 15%, 16%, 17%, 19%, 22%, 25%, 28%, 31%, or 35% conserved in relation to the amino acid sequence identified as SEQ ID NO:2; a conservatively substituted variant thereof; or a functionally equivalent homologue.
Such a synthetic polypeptide can have at least 49 amino acids deleted from the sequence.
The polypeptide can have no cysteine residues or at least two cysteine residues.
The polypeptide can have a molecular weight in the range of from about 1000 to 4000.
In another aspect, the present invention is a first polypeptide having a sequence of amino acids sufficiently duplicative of a second polypeptide which includes an amino acid sequence of any of the synthetic polypeptides, such that the first polypeptide is encoded by a DNA that hybridizes under stringent conditions with DNA encoding the second polypeptide.
The invention includes a chimeric bone stimulating factor comprising an amino acid sequence of any of the synthetic polypeptides.
An agent of the present invention for use in prevention and treatment of a bone reduction related disease can include one or more of the synthetic polypeptides.
As well, a pharmaceutical composition of the present invention for promoting bone growth, can include a therapeutically effective amount of a one or more of the synthetic polypeptides.
The invention includes a method of increasing bone growth in a mammal by administering a therapeutically effective amount of one or more of the synthetic polypeptides.
Such a synthetic polypeptide can be used for the treatment of osteoporosis or to promote bone growth in a mammal.
Such a synthetic polypeptide can be used in the preparation of a medicament for use in promoting bone growth or the treatment of osteoporosis.
The invention includes a diagnostic kit for determining the presence of one or more the synthetic polypeptides, the kit including antibody(ies) to a the polypeptide(s) linked to a reporter system wherein the reporter system produces a detectable response when a predetermined amount of the polypeptide(s) and the antibody(ies) are bound together.
The invention includes an antibody which binds to one or more of the synthetic polypeptides, synthesized using the polypeptide.
Further still, the invention includes an isolated DNA fragment which encodes the expression of any of the synthetic polypeptides, and DNA which differs from the fragment due to the degeneracy of the genetic code. The invention includes a vector which includes such a DNA sequence.
The invention includes a process for producing any of the synthetic polypeptides, which includes:
a) preparing a DNA fragment containing a nucleotide sequence which encodes a polypeptide;
b) incorporating said DNA fragment into an expression vector to obtain a recombinant DNA fragment which contains the DNA fragment and is capable of undergoing replication;
c) transforming a host cell with the recombinant DNA fragment to isolate a transformant which can express the polypeptide; and
d) culturing the transformant to allow the transformant to produce the polypeptide and recovering the polypeptide from resulting cultured mixture.
The invention includes a polypeptide exhibiting bone stimulatory activity in mammals, the polypeptide being up to 65 amino acids in length and having the sequence identified as SEQ ID NO:11, SEQ ID NO:12, or SEQ ID NO:13; analogues thereof wherein the amino acids in the sequence may be substituted, deleted or added, so long as the bone stimulatory activity in mammals derived the three dimensional structure of the sequence is preserved; and conjugates of each of the polypeptides or analogues thereof, wherein if the polypeptide sequence contains a cysteine residue, there are at least two cysteine residues. Such a polypeptide can be substantially pure and the amino acid sequence can have a molecular weight in the range of from about 1000 to about 4000, or from about 1500 to about 3000, or from about 1500 to about 1800. The invention is also a first polypeptide that includes a sequence of amino acids sufficiently duplicative of a second polypeptide having an amino acid sequence corresponding such a polypeptide, or a functionally equivalent homologue thereof, such that the first polypeptide is encoded by a DNA that hybridizes under stringent conditions with DNA encoding the second polypeptide; or a chimeric bone stimulating factor including such an amino acid sequence; or an agent for use in prevention and treatment of a bone reduction related disease which includes such a polypeptide as an active ingredient; or a pharmaceutical composition for promoting bone growth, having a therapeutically effective amount of such a polypeptide; or a method of increasing bone growth in a mammal by administering a therapeutically effective amount of such a polypeptide; having an amino acid sequence of a polypeptide defined in claim 56 or 57; or the use of such a polypeptide for the treatment of osteoporosis or to promote bone growth in a mammal; or the use of such a polypeptide in the preparation of a medicament for use in promoting bone growth or the treatment of osteoporosis; or a diagnostic kit for determining the presence of such a polypeptide, the kit including an antibody to a the polypeptide linked to a reporter system wherein the reporter system produces a detectable response when a predetermined amount of the polypeptide and the antibody are bound together; or an antibody which binds to such a polypeptide, synthesized using the polypeptide; or an isolated DNA fragment which encodes the expression of any such polypeptide, or which differs from the fragment due to the degeneracy of the genetic code; or a vector including a DNA sequence which encodes the expression of any such polypeptide; or a process for producing such a polypeptide, which process includes:
a) preparing a DNA fragment containing a nucleotide sequence which encodes the polypeptide;
b) incorporating the DNA fragment into an expression vector to obtain a recombinant DNA fragment which contains the DNA fragment and is capable of undergoing replication;
c) transforming a host cell with the recombinant DNA fragment to isolate a transformant which can express the polypeptide; and
d) culturing the transformant to allow the transformant to produce the polypeptide and recovering the polypeptide from resulting cultured mixture.
Finally, the invention includes an isolated DNA sequence encoding the amino acid sequence of any of the polypeptides of the invention, or an analogue thereof, wherein the amino acids in the sequence may be substituted, deleted or added, so long as bone stimulatory activity in mammals derived from the three dimensional conformation of the sequence is preserved in a polypeptide comprising the amino acid sequence; sequences which hybridize to the DNA and encode an amino acid sequence of a polypeptide which displays bone stimulatory activity in mammals; and DNA which differs from the sequence due to the degeneracy of the genetic code.